Chapter 17 : Detection Methodology: Pyrosequencing

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Pyrosequencing is a non-gel-based, real-time approach to sequence DNA by monitoring DNA polymerase activity using an enzymatic luminometric inorganic pyrophosphate detection assay. Using pyrosequencing for microbiology identification or drug resistance screening involves three main steps: assay design, PCR, and the actual pyrosequencing. To make pathogen identifications, pyrosequencing assays have frequently used the highly conserved targets that are also used in Sanger sequencing, specifically 16S rRNA in bacteria and mycobacteria and the 18S/internal transcribed spacer (ITS) region in fungi, although other targets are used when the conserved regions are inadequate for species identification. 16S rRNA is a bacterial identification target commonly used by many different technologies, including Sanger sequencing. Pyrosequencing is a real-time sequencing technology alternative to Sanger sequencing. The versatility of pyrosequencing allows bacteria to be identified via 16S single-nucleotide polymorphism (SNP) analysis or de novo sequencing of the variable regions. Pyrosequencing enabled the differentiation of Hantaan virus from Seoul virus, the most common hemorrhagic fever with renal syndrome pathogens in Asia, and of Andes virus from Sin Nombre virus, the two viruses that cause hantavirus cardiopulmonary syndrome in the Americas. Pyrosequencing has been compared prospectively to conventional sequencing for the diagnosis and genotyping of the parasite . Microbial drug resistance is usually developed over time by the introduction of a point mutation or sometimes multiple mutations. The ability to fuse real-time PCR with real-time pyrosequencing may be the catalyst that leads to widespread adoption of pyrosequencing in clinical laboratories.

Citation: Yan L, Slinger R. 2011. Detection Methodology: Pyrosequencing, p 261-273. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch17
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Image of FIGURE 1

Principle of pyrosequencing. (A) Pyrosequencing assay design. PCR primers are located in conserved regions. The biotin-labeled primer is indicated by the solid circle. (B) PCR product. (C) ssDNA is annealed to the sequencing primer at the conserved region. (D) Dideoxynucleotides are added sequentially. An enzymatic cascade reaction generates light signals. (E) The light signals are displayed as a peak in Pyrogram trace. When one nucleotide gets incorporated, a one-peak height light signal is generated. If two identical nucleotides are incorporated, a doublepeak height light signal is generated. The sequence can be read as CTCCT.

Citation: Yan L, Slinger R. 2011. Detection Methodology: Pyrosequencing, p 261-273. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch17
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Image of FIGURE 2

Pyrosequencing built-in control: rifampin resistance testing. (A) Rifampin-susceptible CGCT; (B) rifampin-resistant CGCT.

Citation: Yan L, Slinger R. 2011. Detection Methodology: Pyrosequencing, p 261-273. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch17
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Image of FIGURE 3

Multiplex pyrosequencing. (A) Sequence pattern from A/G SNP in one template. (B) Sequence pattern from T/C SNP in another template. (C) Sequence pattern when both SNPs are analyzed simultaneously.

Citation: Yan L, Slinger R. 2011. Detection Methodology: Pyrosequencing, p 261-273. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch17
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Comparison of Sanger sequencing and pyrosequencing

Citation: Yan L, Slinger R. 2011. Detection Methodology: Pyrosequencing, p 261-273. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch17
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Examples of microorganism identification and typing by pyrosequencing

Citation: Yan L, Slinger R. 2011. Detection Methodology: Pyrosequencing, p 261-273. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch17
Generic image for table

Examples of genotypic antibiotic resistance determination by pyrosequencing

Citation: Yan L, Slinger R. 2011. Detection Methodology: Pyrosequencing, p 261-273. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch17

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